Aberrant activation of Wnt pathways is a common feature of many types of human malignancies. Several studies have demonstrated that the genes encoding secreted Wnt antagonists are frequently silenced in tumor cells by hypermethylation, consistent with the idea that these proteins often function as tumor suppressors. Members of the secreted Frizzled-related protein (sFRP), Dickkopf (Dkk) and Wnt Inhibitory Factor (WIF) families are silenced by hypermethylation in renal cell carcinoma (RCC). Moreover, restoration of sFRP-1 expression in RCC cells markedly inhibited cell proliferation, tumor growth and decreased expression of Wnt target genes, indicating that loss of sFRP-1 is a pivotal event in renal cell carcinogenesis. These studies strongly suggested that detection of the hypermethylaion status of Wnt antagonist genes could serve as a useful biomarker for RCC. This would be especially valuable if hypermethylation of these genes was an early event in the development of RCC, as has been indicated for other tumors, and if a reliable, non-invasive method to recognize gene hypermethylation were available. Evaluation of gene methylation status in circulating DNA has been described, giving credence to its potential use as a powerful screening tool for cancer diagnosis. However, the minute quantities of DNA in circulation and limited sensitivity of conventional polymerase chain reaction (PCR) methodology are impediments to the advancement of this diagnostic modality. We will utilize real-time PCR technology to develop an assay with increased sensitivity and specificity to detect hypermethylated Wnt antagonist genes in serum samples from patients with RCC. This project will take great advantage of access to RCC samples and expertise from collaborators both inside and outside of the NCI community. Development of a successful protocol to detect Wnt antagonist gene hypermethylation could enable early diagnosis and monitoring of RCC, and perhaps also provide guidance in the choice of cancer therapy, all of which would have profound beneficial effects on patient survival. During the past year various technical issues have raised concern about the reliability of the PCR analysis used for detection of hypermethylated Wnt antagonist genes. We validated the detection of methylated DNA corresponding to the genes encoding sFRP-1, sFRP-4, sFRP-5 and Dkk3 using a combination of nested and real-time PCR. However, we have observed a significant amount of irrelevant PCR product that may be interpreted as a false positive signal. By including a melting curve analysis of PCR product, the likelihood of false positive assignments has been reduced. Based on work with methylated sFRP-1 cDNA, it appears that better discrimination between real and false positive signals may be achieved by omitting the nested PCR step and relying on the inherent differences in melting of PCR products generated from bisulfite treated methylated vs. unmethylated DNA (Lorente et al. BMC Cancer 2008).